Loss of C-terminal alpha-helix decreased SDF-1alpha-mediated signaling and chemotaxis without influencing CXCR4 internalization
Abstract
AIM:
To investigate the possibility that a novel alpha-helix-defective mutant of stromal cell-derived factor-1alpha (SDF-1alpha) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4).
METHODS:
According to the genetic sequence of natural SDF-1alpha, a recombinant alpha-helix-defective mutant of SDF-1alpha was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody. Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody.
RESULTS:
Compared with native SDF-1alpha, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation, and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1alpha was confirmed.
CONCLUSION:
Alpha-helix-defective mutant of SDF-1alpha, SDF-1/54R that remained both the N-terminus and the central beta-sheet region, decreased SDF-1alpha-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.
Keywords:
To investigate the possibility that a novel alpha-helix-defective mutant of stromal cell-derived factor-1alpha (SDF-1alpha) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4).
METHODS:
According to the genetic sequence of natural SDF-1alpha, a recombinant alpha-helix-defective mutant of SDF-1alpha was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody. Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody.
RESULTS:
Compared with native SDF-1alpha, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation, and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1alpha was confirmed.
CONCLUSION:
Alpha-helix-defective mutant of SDF-1alpha, SDF-1/54R that remained both the N-terminus and the central beta-sheet region, decreased SDF-1alpha-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.